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KEY POINTS: In mammals, pregnancy complications can trigger an embryonic or fetal origin of cardiac dysfunction. However, underlying mechanisms remain uncertain because the partial contributions of the challenge on the mother, placenta or offspring are difficult to disentangle. The avian embryo permits isolation of the direct effects of suboptimal conditions during development on the cardiac function of the offspring, independent of additional effects on the mother and/or the placenta. Therefore, the objectives of this work were to adapt the isolated Langendorff technique using the chicken embryo to study the physiology of the developing heart. Here, we introduce a novel technique and show the utility of the technique for exploring cardioprotective roles of H2 S in the chicken embryo heart. This work lays the foundation for studying the direct effects of H2 S therapy on the embryonic heart independent of effects on the mother and the placenta in adverse development. ABSTRACT: This study adapted the isolated Langendorff preparation to study the chicken embryo heart in response to ischaemia-reperfusion (IR) injury. The utility of the technique was tested by investigating cardioprotective effects of hydrogen sulphide (H2 S) and underlying mechanisms. Embryonic hearts (19 out of 21 days of incubation) mounted on a Langendorff preparation were exposed to IR (30 min ischaemia) after 4 treatments administered randomly, all as a 1 mm bolus, into the perfusate: saline vehicle (control); sodium hydrogen sulphide (NaHS); NaHS plus glibenclamide, an antagonist of KATP opening (NaHS Glib), and Glib alone (Glib). Relative to controls, NaHS treatment improved cardiac function after ischaemia (mean ± SD for area under the curve, AUC, for left ventricular developed pressure, LVDP: 1767.3 ± 929.5 vs. 492.7 ± 308.1; myocardial contractility, dP/dtmax : 2748.9 ± 1514.9 vs. 763.7 ± 433.1) and decreased infarct size (22.7 ± 8.0 vs. 43.9 ± 4.2%) and cardiac damage (% change in creatinine kinase, 49.3 ± 41.3 vs. 214.6 ± 155.1; all P < 0.05). Beneficial effects of NaHS were blocked by Glib. Glib alone had no effects. NaHS increased coronary flow rate (CFR) during baseline (mean ± SD for AUC: 134.3 ± 91.6 vs. 92.2 ± 35.8) and post IR (1467 ± 529.5 vs. 748.0 ± 222.1; both P < 0.05). However, this effect was not prevented by Glib. Therefore, the chicken embryo heart is amenable for study via the Langendorff preparation under basal conditions and during IR. The data show that H2 S confers embryonic cardiac protection via opening of myocardial KATP channels and not via increasing CFR. H2 S may prove a useful therapeutic agent to protect the human fetal heart against IR injury, as may occur in complicated labour.